The present invention is a novel selectable and autonomously replicating recombinant DNA expression vector which contains 1) a transcriptional and translational activating nucleotide sequence, 2) a gene that codes for a bioactive derivative of bovine growth hormone (bGH) and 3) a replicon which, under induced conditions, loses copy number control. The invention further contains novel transformants of the aforementioned vectors, bovine growth hormone derivatives and methods of use.
The development and exploitation of recombinant DNA technology for the production of bGH has been severely handicapped by problems of gene expression. Some of these problems, detailed in European Patent Application Publication No. 0075444, involve the transcription of functionally suboptimal mRNA.
The development and exploitation of recombinant DNA technology has been limited by the general paucity of vectors providing for high levels of gene expression. Although the lac (Roberts et al., 1979, Proc. Nat. Acad. Sci. USA 76:5596 and Guarante et al., 1980, Cell 20:543), trp (Hallewell and Emtage, 1980, Gene 9:27), bacteriophage .lambda. P.sub.L (Remaut et al., 1981, Gene 15(1):81, Bernard et al., 1979, Gene 5:59 and Derom et al., 1982, Gene 17:45) and lpp (Ziebel et al., 1981, J. Bacteriol. 145:654, Lee et al., 1981, J. Bacteriol. 146:861 and Nakamura and Inouye, 1979, Cell 18:1109) promoter systems have been incorporated into assorted recombinant DNA vectors, few, if any, other expression systems are available. In fact, many heterologous genes are not expressed or are, at best, only poorly expressed using these known expression vectors. Such failure of expression is frequently associated with the transcription of a mRNA that is sub-optimal or incapable of binding to ribosomes. This may be due to the formation of inappropriate stem and loop structures that sequester sites for ribosome binding or to the presence of sequences that inhibit ribosome binding or initiation of translation. Some of these problems are further described in European Patent Application Publication No. 0075444.
The present invention solves problems of expression without requiring the synthetic modification of a gene. Such synthetic modification is not only time consuming and costly but also substantially increases the risk of accidentally introducing errors into a DNA sequence. This problem is circumvented in the present invention by altering the 5' end of a mRNA transcript without modifying or changing the sequence of a particular gene of interest.
For purposes of the present invention as disclosed and claimed herein, the following terms are as defined below.
Recombinant DNA Cloning Vector--any autonomously replicating agent, including but not limited to plasmids, containing a DNA molecule to which one or more additional DNA segments can or have been added.
Recombinant DNA Expression Vector--any recombinant DNA cloning vector into which one or more transcriptional and translational activator sequence(s) have been incorporated.
Transcriptional Activating Sequence--any DNA sequence that directs or provides for the transcription of DNA into a mRNA transcript.
Translational Activating Sequence--any DNA sequence that directs or provides for the translation of a mRNA transcript into a peptide, polypeptide or protein.
Translational Start Signal--any DNA triplet that codes for a translational start codon.
Translational Stop Signal--any DNA triplet that codes for a translational stop codon.
Transformation--the introduction of DNA into a recipient host cell.
Transformant--a recipient host cell that has undergone transformation.
Restriction Fragment--any linear DNA sequence generated by the action of one or more restriction enzymes.
Functional Polypeptide--a recoverable biologically active homologous or heterologous polypeptide or precursor, a recoverable polypeptide containing a heterologous polypeptide and a portion or whole of a homologous polypeptide, or a recoverable bio-inactive fusion polypeptide containing a heterologous polypeptide and a bio-inactivating polypeptide which can be specifically cleaved.
Fused Gene Product--a recoverable heterologous polypeptide which contains a portion or whole of a homologous polypeptide.
Replicon--any DNA sequence that controls the replication of recombinant DNA cloning and expression vectors.
Runaway Replicon--a replicon which lacks or can be induced to lose copy number control, such loss resulting in the uncontrolled replication and an extreme increase in the copy number of DNA into which such replicon has been incorporated.